Project Summary The goal of this project is to develop a novel probiotic-based therapeutic for the treatment of inflammatory bowel disease (IBD) that is ideal for long-term maintenance treatment. Over 1.6 million adults and children in the United States suffer from IBD, an umbrella term encompassing two chronic inflammatory diseases of the gastrointestinal tract: Crohn's disease (CD) and ulcerative colitis (UC)1. IBD is typically diagnosed in the second or third decades of life, it is life-long, and there is no cure. IBD is treated most commonly with systemic immune suppressants or cytokine (e.g. TNF, IL-12/23) blockers that can have serious side-effect profiles, especially when used long-term. Thus, new, safer alternatives, particularly for long-term maintenance therapy, are needed and would be life-changing for patients suffering from these difficult gastrointestinal diseases. Intestinal immune regulatory signals tightly govern healthy gut homeostasis. Breakdown of such regulatory mechanisms may result in IBD (e.g., CD, UC)2. The human microbiome, harboring trillions of bacteria, is a critical regulator of these immune mechanisms. Commensal bacteria function to maintain integrity of the intestinal epithelial barrier, as well as regulate innate and adaptive immune cell function and immune homeostasis. One common commensal bacterium, Lactobacillus acidophilus, contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA)3-6. These Slps interact with pattern recognition receptors (PRR; e.g., C-type lectin receptors) expressed on innate immune intestinal cells to mobilize fine-tuned immunity in steady state and disease conditions5. Recently, our research team demonstrated that SlpA binding to the C-type lectin Specific Intracellular adhesion molecule-3 Grabbing Non-integrin homolog-Related 3 (SIGNR3) receptor expressed on dendritic cells lining the gut prevents experimentally induced colitis in multiple models7. Oral delivery of SlpA reduced inflammatory cytokines, strengthened the mucosal membrane barrier, and supported a healthier microbiota make-up in animal models of gut inflammation. In contrast, these effects and protection was not observed in Signr3-/- mice, suggesting that SlpA interaction with SIGNR3 plays a key protective role in regulating the disease condition7. Our goal is to develop R-3750, a novel Lactococcus (L.) lactis probiotic recombinantly expressing SlpA that will carry this SlpA biological targeting agent to the intestinal space and function via natural gut immune- microbiome pathways to induce protective regulatory signals in innate cells (e.g., dendritic cells, etc.). R-3750 will reduce gut inflammation maintaining gastrointestinal mucosal barrier function and promoting more normal microbiota homeostasis. The specific aims are to: 1) generate L. lactis clones recombinantly expressing SlpA from a genome-integrated operon, 2) select a R-3750 clinical candidate based on functional screening, growth rate, SlpA expression, and stability, and 3) demonstrate activity in the murine T cell induced colitis model. Successful commercialization of R-3750 will provide a profound medical advancement in the treatment of IBD.